Asad Jan

OsTZF1, a CCCH-Tandem Zinc Finger Protein, Confers Delayed Senescence and Stress Tolerance in Rice by Regulating Stress-Related Genes

OsTZF1, a CCCH-type zinc finger protein, acts as a negative regulator of leaf senescence in rice under stress conditions and confers abiotic stress tolerance by delaying stress-response phenotypes, possibly through the control of RNA metabolism of stress-responsive genes. OsTZF1 is a member of the CCCH-type zinc finger gene family in rice (Oryza sativa). Expression of OsTZF1 was induced by drought, high-salt stress, and hydrogen peroxide. OsTZF1 gene expression was also induced by abscisic acid, methyl jasmonate, and salicylic acid. Histochemical activity of β-glucuronidase in transgenic rice plants containing the promoter of OsTZF1 fused with β-glucuronidase was observed in callus, coleoptile, young leaf, and panicle tissues. Upon stress, OsTZF1-green fluorescent protein localization was observed in the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF1 driven by a maize (Zea mays) ubiquitin promoter (Ubi:OsTZF1-OX [for overexpression]) exhibited delayed seed germination, growth retardation at the seedling stage, and delayed leaf senescence. RNA interference (RNAi) knocked-down plants (OsTZF1-RNAi) showed early seed germination, enhanced seedling growth, and early leaf senescence compared with controls. Ubi:OsTZF1-OX plants showed improved tolerance to high-salt and drought stresses and vice versa for OsTZF1-RNAi plants. Microarray analysis revealed that genes related to stress, reactive oxygen species homeostasis, and metal homeostasis were regulated in the Ubi:OsTZF1-OX plants. RNA-binding assays indicated that OsTZF1 binds to U-rich regions in the 3′ untranslated region of messenger RNAs, suggesting that OsTZF1 might be associated with RNA metabolism of stress-responsive genes. OsTZF1 may serve as a useful biotechnological tool for the improvement of stress tolerance in various plants through the control of RNA metabolism of stress-responsive genes.

Comparative functional analysis of six drought-responsive promoters in transgenic rice

Rice production is greatly affected by environmental stresses such as drought and high salinity. Transgenic rice plants tolerant to such stresses are expected to be produced. Stress-responsive promoters with low expression under normal growth conditions are needed to minimize the adverse effects of stress-tolerance genes on rice growth. We performed expression analyses of drought-responsive genes in rice plants using a microarray, and selected LIP9, OsNAC6, OsLEA14a, OsRAB16D, OsLEA3-1, and Oshox24 for promoter analysis. Transient assays using the promoters indicated that AREB/ABF (abscisic acid (ABA)-responsive element-binding protein/ABA-binding factor) transcription factors enhanced expressions of these genes. We generated transgenic rice plants containing each promoter and the β-glucuronidase (GUS) reporter gene. GUS assays revealed that the LIP9 and OsNAC6 promoters were induced by drought, high salinity, and ABA treatment, and both promoters showed strong activity under normal growth conditions in the root. The other promoters were strongly induced by stresses and ABA, but showed low activity under normal growth conditions. In seeds, GUS staining showed that Oshox24 expression was low and expressions of the other genes were high. Transgenic rice plants overexpressing OsNAC6 under the control of the Oshox24 promoter showed increased tolerance to drought and high salinity, and no growth defects. These data suggest that the Oshox24 promoter is useful to overexpress stress-tolerance genes without adversely affecting growth.

Identification of phosphoproteins regulated by gibberellin in rice leaf sheath

To identify the gibberellin (GA) signaling components involved in rice leaf sheath elongation process, protein phosphorylation changed by GA3 was analyzed. The protein kinase activities in rice leaf sheath were assessed in an in-gel kinase assay using SDS-polyacrylamide gel containing histone III-S as a substrate. The activity of a putative 54-kDa calcium dependent protein kinase (CDPK) in cytosolic fraction in rice leaf sheath increased significantly by GA3. Further, phosphorylation status of the proteins changed by GA3 in rice leaf sheath were detected by in vitro protein phosphorylation followed by two-dimensional polyacrylamide gel electrophoresis and the phosphoproteins were identified by mass spectrometry. Sixty phosphoproteins was detected after in vitro protein phosphorylation and the phosphorylation of 7 proteins was enhanced by GA3 treatment. The addition of GA3 treated cytosolic fraction of leaf sheath further increased the phosphorylation of 4 proteins, glyoxalase-I, cytoplasmic malate dehydrogenase, glyceraldehydes-3-phosphate dehydrogenase and another unknown protein. The protein kinase inhibitor, staurosporine inhibited the phosphorylation of these proteins in vitro. Other hormones, particularly, indole acetic acid, 6-benzylaminopurine and abscisic acid did not change the phosphorylation status of these proteins. The identified proteins did not show any change by GA3 treatment at transcription level. The abundance of glyoxalase-I and cytoplasmic malate dehydrogenase remained unchanged by GA3 treatment as detected on 2D-gel by silver staining, unlike for glyceraldehydes-3-phosphate dehydrogenase. Results suggest that the phosphoproteins, glyoxalase-I and cytoplasmic malate dehydrogenase in rice leaf sheath could be important signaling components of GA3, downstream to 54-kDa CDPK.

Mutation of alpha-tubulin genes in trifluralin-resistant water foxtail (Alopecurus aequalis).

BACKGROUND Trifluralin-resistant biotypes of water foxtail (Alopecurus aequalis) have been identified in wheat fields from northern Kyushu, Japan. Water foxtail is a winter-annual grassy weed, causing substantial crop losses. This study reports on mutation in α-tubulin (TUA) genes from water foxtail, the site of action of trifluralin. RESULTS Two trifluralin-sensitive (S) Chikugo and Ukiha biotypes and four trifluralin-resistant (R) Asakura-1, Asakura-2, Tamana and Tosu biotypes of water foxtail were used for herbicide resistance analysis. R biotypes showed 5.7-30.7-fold trifluralin resistance compared with the S biotypes. No differences in the uptake and translocation of (14)C-trifluralin were observed between Chikugo (S) biotype and Asakura-1 (R) biotype. Most of the (14)C detected in the plant material was in the root tissue, and no substantial increases were noted in shoot tissues. Comparative TUA sequence analysis revealed two independent single amino acid changes: change of Val into Phe at position 202 in TUA1 and change of Leu into Met at position 125 in TUA3 in Asakura-1 biotype. In the Tamana (R) biotype, two amino acid changes of Leu to Phe at position 136 and Val to Phe at position 202 were observed in the predicted amino acid sequence of TUA1, compared with Chikugo (S) biotype. CONCLUSION The results provide preliminary molecular explanation for the resistance of water foxtail to trifluralin, a phenomenon that has arisen as a result of repeated exposure to this class of herbicide. This is the first report of α-tubulin mutation in water foxtail and for any Alopecurus species reported in the literature.

Characterization of a histidine- and alanine-rich protein showing interaction with calreticulin in rice

Calreticulin (CRT) is a major calcium-sequestering protein in the endoplasmic reticulum and has been implicated in a variety of cellular functions. To analyze the function of CRT in rice, a yeast two-hybrid protein interaction assay was used for identifying interacting proteins. Fourteen of 17 interacting cDNA clones found coded for a novel histidine- and alanine-rich protein (OsHARP) of 342 amino acid residues. The mRNA expression level of OsHARP was up-regulated in rice seedlings treated with gibberellin (GA), but not ABA and showed a similar pattern as OsCRT mRNA. Rice plants transformed with the OsHARP promoter-GUS construct showed GUS staining in the basal parts of leaf sheaths, and although GUS activity increased when treated with GA3, it was not as high an increase as when mRNA was analyzed. To elucidate the role of OsHARP in leaf sheath elongation, antisense OsHARP transgenic rice lines were constructed. The antisense OsHARP transgenic rice plants were consistently shorter than the vector control under normal conditions. To examine whether OsHARP expression would affect other proteins, basal leaf sheaths from antisense OsHARP transgenic rice plants were analyzed using proteomic techniques. In antisense transgenic-rice OsHARP plants, OsCRT was down-regulated and the levels of 20 other proteins were changed compared to the pattern of the vector control. These results signify an important role of HARP in rice leaf sheath cell division or elongation and suggest that CRT may interact with HARP during certain stages of development.

Functional Characterization of Gibberellin-Regulated Genes in Rice Using Microarray System

Gibberellin (GA) is collectively referred to a group of diterpenoid acids, some of which act as plant hormones and are essential for normal plant growth and development. DNA microarray technology has become the standard tool for the parallel quantification of large numbers of messenger RNA transcripts. The power of this approach has been demonstrated in dissecting plant physiology and development, and in unraveling the underlying cellular signaling pathways. To understand the molecular mechanism by which GA regulates the growth and development of plants, with reference to the monocot model plant—rice, it is essential to identify and analyze more genes and their products at the transcription and translation levels that are regulated by GA. With the availability of draft sequences of two major rice types, indica and japonica rice, it has become possible to analyze global expression profiles of genes on a genome scale. In this review, the progress made in finding new genes in rice leaf sheath using microarray system and their characterization is discussed. It is believed that the findings made in this regard have important implications for understanding the mechanism by which GA regulates the growth and development of rice.

Impact of Transgenic Brassica napus Harboring the Antifungal Synthetic Chitinase (NiC) Gene on Rhizosphere Microbial Diversity and Enzyme Activities

Transgenic Brassica napus harboring the synthetic chitinase (NiC) gene exhibits broad-spectrum antifungal resistance. As the rhizosphere microorganisms play an important role in element cycling and nutrient transformation, therefore, biosafety assessment of NiC containing transgenic plants on soil ecosystem is a regulatory requirement. The current study is designed to evaluate the impact of NiC gene on the rhizosphere enzyme activities and microbial community structure. The transgenic lines with the synthetic chitinase gene (NiC) showed resistance to Alternaria brassicicola, a common disease causing fungal pathogen. The rhizosphere enzyme analysis showed no significant difference in the activities of fivesoil enzymes: alkalyine phosphomonoestarase, arylsulphatase, β-glucosidase, urease and sucrase between the transgenic and non-transgenic lines of B. napus varieties, Durr-e-NIFA (DN) and Abasyne-95 (AB-95). However, varietal differences were observed based on the analysis of molecular variance. Some individual enzymes were significantly different in the transgenic lines from those of non-transgenic but the results were not reproducible in the second trail and thus were considered as environmental effect. Genotypic diversity of soil microbes through 16S–23S rRNA intergenic spacer region amplification was conducted to evaluate the potential impact of the transgene. No significant diversity (4% for bacteria and 12% for fungal) between soil microbes of NiC B. napus and the non-transgenic lines was found. However, significant varietal differences were observed between DN and AB-95 with 79% for bacterial and 54% for fungal diversity. We conclude that the NiC B. napus lines may not affect the microbial enzyme activities and community structure of the rhizosphere soil. Varietal differences might be responsible for minor changes in the tested parameters.